Materials and methods for protein production

Abstract

This document relates to materials and methods for the production of protein. In one aspect, this document provides a nucleic acid construct including a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element includes a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28.

What is claimed is: 
     
       1. A nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises one or more mutations at a nucleotide position selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28. 
     
     
       2. The nucleic acid construct of  claim 1 , wherein the first alcohol oxidase promoter element comprises one or more mutations at a nucleotide position selected from the group consisting of nucleotide positions corresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO: 28. 
     
     
       3. The nucleic acid construct of  claim 1 , wherein the first alcohol oxidase promoter element comprises mutations at nucleotide positions corresponding to T688, A696, T702, A712, and T714 relative to SEQ ID NO: 28. 
     
     
       4. The nucleic acid construct of  claim 1 , wherein the first alcohol oxidase promoter element comprises one or more mutations selected from the group consisting of mutations corresponding to T146C, C154T, T303C, T426A, A433T, A435G, T530A, C572T, T596C, T617C, T688C, A696T, T702C, A709G, A712G, T714G, A790G, A841T, and T862A relative to SEQ ID NO: 28. 
     
     
       5. The nucleic acid construct of  claim 1 , wherein the first alcohol oxidase promoter element comprises one or more mutations selected from the group consisting of T688C, A696T, T702C, A712G, and T714G relative to SEQ ID NO: 28. 
     
     
       6. The nucleic acid construct of  claim 1 , wherein the first alcohol oxidase promoter element is an alcohol oxidase 1 promoter element. 
     
     
       7. The nucleic acid construct of  claim 1 , further comprising a nucleotide sequence, wherein the nucleotide sequence is operably linked to the first alcohol oxidase promoter element. 
     
     
       8. The nucleic acid construct of  claim 7 , wherein the nucleotide sequence encodes a first protein. 
     
     
       9. The nucleic acid construct of  claim 8 , wherein the first protein is selected from the group consisting of an antibody or fragment thereof, an enzyme, a regulatory protein, a peptide hormone, a blood clotting protein, a cytokine, a cytokine inhibitor, and a heme-binding protein. 
     
     
       10. The nucleic acid construct of  claim 8 , wherein the first protein is a heme-binding protein. 
     
     
       11. A cell comprising a first nucleic acid construct, wherein the first nucleic acid construct is the nucleic acid construct of  claim 1 . 
     
     
       12. A method of producing a product in a cell comprising:
 expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises one or more mutations at a nucleotide position selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28. 
 
     
     
       13. The method of  claim 12 , wherein the first alcohol oxidase promoter element includes one or more mutations at a nucleotide position selected from the group consisting of nucleotide positions corresponding to of T688, A696, T702, A712, and T714 relative to SEQ ID NO: 28. 
     
     
       14. The method of  claim 12 , wherein the first alcohol oxidase promoter element is an alcohol oxidase 1 promoter element. 
     
     
       15. The method of  claim 12 , wherein the nucleotide sequence operably linked to the first alcohol oxidase promoter element encodes a first protein. 
     
     
       16. The method of  claim 12 , wherein the method is carried out in the absence of added methanol.